A stability-indicating high-performance liquid chromatography-mass spectrometry times 2 method was developed to establish the stability of acetyl-l-carnitine dissolved in 5% dextrose in water; quantitation of acetyl-l-carnitine and its hydrolysis product I-carnitine was performed using this method. Acetyl-l-carnitine dissolved in water was stress-degraded at a pH range of 3 to 12, and conversion to l-carnitine was quantified over 18 hours. The method was further validated by stressing the acetyl-l-carnitine solution at 68 degrees C, 82 degrees C, and 90 degrees C for up to 10 days, yielding a temperature-dependent hydrolysis rate constant. Acetyl-l-carnitine solutions were stored at 25 degrees C and 4 degrees C to 8 degrees C for 33 days to validate the kinetics prediction. The liquid chromatography-mass spectrometry times 2 method was sensitive and specific, allowing rapid separation and simultaneous quantitation of acetyl-l-carnitine and l-carnitine. Acetyl-l-carnitine dissolved in aqueous solutions is stable at neutral to acidic pH, but unstable at pH > 9. After 1 hour storage at room temperature, only 72.6% of acetyl-l-carnitine was left at pH 11 and 4.2% left at pH 12. The kinetics relationship between temperature and rate constant was In(k) = -8650.1 /T + 20.344 (r2 = 0.9851) at pH 5.2. The time required to degrade 15% of acetyl-I-carnitine was estimated to be 38 days at 25 degrees C or 234 days at 8 degrees C, and was confirmed with actual storage stability testing. Acetyl-l-carnitine dissolved in water (pH 5.2) at concentrations of 1 and 10 mg/mL was found stable at room temperature or refrigerated for at least 33 days using the established stability-indicating method. Acetyl-l-carnitine solutions are not stable at basic pH. When reconstituted in water, acetyl-l-carnitine is stable for over 30 days at room temperature or under refrigeration.